Full affinity mapping of the Human PDZ-PBM interactome and interactome-phenotype relationship of the viral HPV oncoprotein E6. – Full-PDZ-PBM-HPVE6

Many protein-protein interactions boil down to domain-motif interactomes. For instance, in human, the 266 PDZ domains (the PDZome) and their 4000 putative C-terminal PBMs (PDZ-Binding Motifs) mostly situated at the C-termini of partner proteins define a network of one million potential interactions, quantitatively defined by affinity constants (KD). The PDZ-PBM network is often hijacked by viral proteins bearing their own PBMs. Here, we will demonstrate the proof of concept of interactome-wide affinity mapping, and perform the first ever quantitative interactome-phenotype relationship study, for a model viral oncoprotein. We will experimentally measure the 10^6 Kd values of all the human PDZ-PBM pairs and of an additional subset of PDZ-PBM interactions engaged by "viral hijacker PBMs", using high-throughput holdup assay complemented by fluorescence polarization assays and affinity purification-mass spectrometry experiments on human cell line extracts. We will make the data accessible on a dedicated server, and analyze by bioinformatics diverse aspects of the resulting multidimensional interactomic space, where all interactants, including the viral intruders, are positioned according to their binding properties. Finally, we will explore the quantitative interactome-phenotype relationship of the HPV E6 oncoprotein involved in various human cancers. To this aim, we will (i) systematically measure the PDZome-binding affinities of a series of mutants of the C-terminal PBM of E6, (ii) measure and compare the full transcriptomes and proteomes of cells stably expressing various constructs of E6 bearing the same mutants of the PBM, and (iii) investigate potential interactome-transcriptome-proteome correlations. Both the fully explored PDZ-PBM human interactome and the interactome-phenotype relationship study will represent breakthroughs in quantitative system biology, opening the way to countless studies following the same principles.